Prediction of peanut allergy in adolescence by early childhood storage protein-specific IgE signatures: the BAMSE population-based birth cohort

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1 Accepted Manuscript Prediction of peanut allergy in adolescence by early childhood storage protein-specific IgE signatures: the BAMSE population-based birth cohort Anna Asarnoj, MD, PhD, Carl Hamsten, PhD, Christian Lupinek, MD, PhD, Erik Melén, MD, PhD, Niklas Andersson, MSc, Josep M. Anto, MD, PhD, Jean Bousquet, MD, PhD, Rudolf Valenta, MD, Marianne van Hage, MD, PhD, Magnus Wickman, MD, PhD PII: S (17)30221-X DOI: /j.jaci Reference: YMAI To appear in: Journal of Allergy and Clinical Immunology Received Date: 21 June 2016 Revised Date: 12 December 2016 Accepted Date: 19 December 2016 Please cite this article as: Asarnoj A, Hamsten C, Lupinek C, Melén E, Andersson N, Anto JM, Bousquet J, Valenta R, van Hage M, Wickman M, Prediction of peanut allergy in adolescence by early childhood storage protein-specific IgE signatures: the BAMSE population-based birth cohort, Journal of Allergy and Clinical Immunology (2017), doi: /j.jaci This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Asarnoj et al Prediction of peanut allergy in adolescence by early childhood storage protein-specific IgE signatures: the BAMSE population-based birth cohort Anna Asarnoj, MD, PhD 1,2, Carl Hamsten*, PhD 1, Christian Lupinek*, MD, PhD 3, Erik Melén, MD, PhD 4,5, Niklas Andersson, MSc 4, Josep M Anto, MD, PhD 6,7,8,9, Jean Bousquet, MD, PhD 10, Rudolf Valenta, MD 3, Marianne van Hage, MD, PhD 1,, Magnus Wickman, MD, PhD 4,5,11 * shared second authorship shared last authorship 1. Immunology and Allergy Unit, Department of Medicine, Karolinska Institutet, and Karolinska University Hospital, Stockholm, Sweden 2. Astrid Lindgren Children s Hospital, Karolinska University Hospital, Stockholm, Sweden 3. Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Austria 4. Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 5. Dept. of Pediatrics, Sachs Children s Hospital, Stockholm, Sweden 6. ISGlobal, Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain 7. IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain 8. Universitat Pompeu Fabra (UPF), Barcelona, Spain 9. CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain 10. University Hospital, Montpellier, France; INSERM, VIMA : Ageing and chronic diseases. Epidemiological and public health approaches, U1168, Paris; UVSQ, UMR-S 1168, Université Versailles St- Quentin-en-Yvelines, France Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden 1

3 Asarnoj et al Short title: Evolution of peanut allergy during childhood Corresponding author: Anna Asarnoj, MD, PhD Immunology and Allergy Unit, L2:04, Department of Medicine Karolinska Institutet and University Hospital S Stockholm, Sweden Telephone: Fax: Key words Peanut allergy; allergen; Ara h 2; component resolved diagnostics; IgE; molecular allergology; sensitization; birth cohort; BAMSE Funding Supported by the Swedish Asthma and Allergy Research Foundation; Stockholm County Council; the Swedish Research Council of Health, Working Life and Welfare; the Swedish Research Council, the Swedish Society of Medicine, the Swedish Heart-Lung Foundation; the Swedish Cancer and Allergy Foundation; the Konsul Th Berg s Foundation, the Magnus Bergvall Foundation, the Swedish Association for Allergology, the European Commission s Seventh Framework 29 Program MeDALL under grant agreement no , and in part by grant F4605 of the Austrian Science Fund (FWF). 50 2

4 Asarnoj et al Author Contributions Data acquisition: AA, CL, EM, NA, RV, MvH, MW; Technical support: CH, CL, NA, RV; Material support: CL, RV, MvH; Manuscript writing: AA, CH, MvH, MW; Critical revision of the manuscript: EM, JA, JB, RV; Study supervision: EM, MvH, MW 3

5 Asarnoj et al Abbreviations Ara h: Arachis hypogaea PR-10: Pathogenesis-related protein family 10 LTP: Lipid transfer protein 95% CI: 95% Confidence interval OAS: Oral allergy syndrome DBPCFC: Double-blind placebo-controlled food challenge 4

6 Asarnoj et al Capsule summary (34 words) IgE signatures to peanut allergen measured in early childhood allow predicting the likelihood of peanut allergy in adolescence. Peanut symptoms and Ara h 2 IgE >2.0 ISU-E at 4 years predict peanut allergy in adolescence. 5

7 Asarnoj et al 71 To the Editor: One of the most frequent and severe forms of food allergy is caused by peanuts 1. IgE reactivity to peanut storage proteins, in particular to Ara h 2, is associated with systemic reactions 2. However, in some regions the lipid transfer protein Ara h 9 is an important allergen molecule whereas in birch endemic areas, the PR-10 protein Ara h 8 is a more common cross-reactive component 3. In the Isle of Wight birth cohort, peanut allergy showed an early onset 4. Interestingly, the percentage of symptomatic patients did not increase but an increase in peanut extract sensitization was noted until adolescence 4. The development of IgE antibody reactivity to the different peanut allergen molecules as well as how IgE sensitization to these molecules contributes to development of peanut symptoms has not yet been explored. In this study, the evolution of sensitization to peanut allergen molecules from early childhood to adolescence was investigated for the first time. We used a random subset of 778 children from the Swedish BAMSE birth cohort (N=4089) 5 where complete relevant questionnaire data from baseline (2 months), 1, 2, 4, 8 and 16 years and blood samples from 4, 8 and 16 years were available. For some of the analyses, this population-based study group was enriched with additional 84 peanut extract-sensitized children (at 4, 8 and/or 16 years) from the same cohort. Thus, our peanut-enriched study group included 862 children (778 from the populationbased study group enriched with 84 peanut sensitized children) (Figure E1). Serum samples were analyzed for IgE antibodies to peanut extract, and at 8 years of age also to Ara h 2, by ImmunoCAP (Thermo Fisher AB, Uppsala, Sweden) and for peanut allergen molecules (Ara h 1, h 2, h 3, h 6, h 8 and h 9) using a modified allergen chip based on ISAC technology (Thermo Fisher) 5. The correlation factor (rho) between the IgE levels to Ara h 2 at 8 years measured with microarray and ImmunoCAP was high: A linear regression with ImmunoCAP and microarray Ara h 2 IgE levels was performed to calculate corresponding values. Detailed information on methods and statistics is provided in the Online Repository at In the population-based study group, the frequencies of sensitization to storage proteins showed little change from 4 to 16 years, but sensitization to the birch pollen homologous allergen molecule Ara h 8 increased at each time point (Figures 1A and 1B). Any reported systemic symptom to peanut in combination with peanut storage protein sensitization increased from 1.4% at 8 years to 3.0% at 16 6

8 Asarnoj et al years (p=0.03, Table E1). The prevalence rates and trajectories, i.e. onset, persistence and transition of IgE reactivity, as well as IgE-levels to peanut extract and the different peanut allergen molecules are displayed in Figures 1A and 1B. Due to the substantial overlap of sensitization to the peanut storage proteins (Figures E2 and E3), no multivariate logistic regression estimates for IgE reactivity to each storage protein in relation to allergic symptoms to peanut could be performed. Of the 54 Ara h 2-sensitized children at 16 years in the peanutenriched study group, 45 (83%) were already sensitized at 4 years and 49 (91%) reported symptoms to peanut at 16 years. Only one participant had de novo Ara h 2 sensitization (0.8 ISU-E) after 8 years (data not shown). The high rate of storage protein sensitization at 4 years among children developing peanut allergy during the first 16 years of life raises the important question of prevention. In children with both peanut and birch pollen sensitization, one fraction was mono-sensitized to Ara h 8 (Figure 2). These children reported no peanut symptoms or only oral allergy syndrome (OAS) at 16 years of age. We have previously reported that sensitization to Ara h 8 is associated with mild OAS or peanut tolerance 6. Interestingly in this study, most children with onset of peanut sensitization after 4 years had IgE reactivity to Ara h 8 only and not to the storage peanut proteins, and were mostly asymptomatic or without report of any systemic symptoms 4, 6. Another fraction of children was sensitized to Ara h 2 (regardless co-sensitization to Ara h 8). At 16 years, peanut symptoms at exposure were reported in 91% of these Ara h 2 sensitized participants. The predicted longitudinal likelihood of reporting peanut allergy at 16 years in the peanut-enriched study group (n=862) was plotted in relation to IgE-levels to Ara h 2 (Figure E4A, incident symptoms Figure E4B), peanut extract (Figure E4C) as well as to the number of sensitizing peanut allergen molecules 4 years of age (Figure E4D). An IgE-level of >2.0 ISU-E to Ara h 2, >15.5 ku A /L to peanut extract or IgE reactivity ( 0.3 ISU-E) to at least 4 peanut allergen molecules at 4 years of age corresponded to a 95% likelihood of reporting peanut symptoms 12 years later. Due to the large number of children investigated in the BAMSE cohort it was not possible to verify peanut symptoms with the double-blind placebo-controlled food challenge (DBPCFC). However, the calculated longitudinal peanut extract-specific IgE level at 4 years (15.5 ku A /L) with a 95% likelihood of peanut symptoms at 16 years is very similar to the one reported in a cross-sectional clinical study by Sampson and colleagues (15 ku A /L) where DBPCFC were included 7. Several previous DBPCFC studies 7

9 Asarnoj et al demonstrate that even low Ara h 2 IgE-levels are associated with peanut allergy in almost all cases, which was shown in our study as well although most other studies measured Ara h 2 IgE with the ImmunoCAP method 2, 7, 8. As the results in ISAC are semiquantitative and may be influenced by blocking IgG antibodies, they cannot simply be transferred to ImmunoCAP. We made a comparison with ImmunoCAP values at 8 years of age and in the BAMSE cohort, an Ara h 2 level of 2.0 ISU-E in microarray corresponded to 4.1 ku/l in ImmunoCAP (data not shown). In summary, our study is the first to examine the onset and persistence of IgE sensitization to peanut allergen molecules in relation to peanut symptoms from preschool age to adolescence. We identified two distinct phenotypes in the development of peanut allergy from childhood to adolescence. One phenotype develops early in life and is related to Ara h 2 sensitization and risk of systemic reactions after peanut exposure. Children with this phenotype rarely outgrow their symptoms. The second phenotype, which starts later in childhood, is related to Ara h 8 sensitization and non-systemic reactions after peanut ingestion. Furthermore, we show that in preschool children, Ara h 2 sensitization or polysensitization to peanut storage proteins are superior in predicting peanut allergy in adolescence compared to peanut extract sensitization. We suggest that measuring IgE to peanut allergen molecules may help clinicians improve the diagnosis and prognosis of peanut allergy. Acknowledgements We would like to thank all participating families and the staff in the BAMSE study as well as the staff at the laboratory of Professor Rudolf Valenta. Thermo Fisher Scientific kindly provided the ImmunoCAP singleplex reagents for the study. Anna Asarnoj, MD, PhD Carl Hamsten*, PhD Christian Lupinek*, MD, PhD Erik Melén, MD, PhD Niklas Andersson, MSc Josep M Anto, MD, PhD 8

10 Asarnoj et al Jean Bousquet, MD, PhD Rudolf Valenta, MD Marianne van Hage, MD, PhD Magnus Wickman, MD, PhD on behalf of the MeDALL consortium * shared second authorship shared last authorship 9

11 Asarnoj et al References 1. Valenta R, Hochwallner H, Linhart B, Pahr S. Food allergies: the basics. Gastroenterology 2015; 148: e4. 2. Nicolaou N, Murray C, Belgrave D, Poorafshar M, Simpson A, Custovic A. Quantification of specific IgE to whole peanut extract and peanut components in prediction of peanut allergy. J Allergy Clin Immunol 2011; 127: Vereda A, van Hage M, Ahlstedt S, Ibanez MD, Cuesta-Herranz J, van Odijk J, et al. Peanut allergy: Clinical and immunologic differences among patients from 3 different geographic regions. J Allergy Clin Immunol 2011; 127: Arshad SH, Venter C, Roberts G, Dean T, Kurukulaaratchy R. The natural history of peanut sensitization and allergy in a birth cohort. J Allergy Clin Immunol 2014; 134: e6. 5. Lupinek C, Wollmann E, Baar A, Banerjee S, Breiteneder H, Broecker BM, et al. Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergenchip. Methods 2014; 66: Asarnoj A, Nilsson C, Lidholm J, Glaumann S, Ostblom E, Hedlin G, et al. Peanut component Ara h 8 sensitization and tolerance to peanut. J Allergy Clin Immunol 2012; 130: Sampson HA. Utility of food-specific IgE concentrations in predicting symptomatic food allergy. J Allergy Clin Immunol 2001; 107: Eller E, Bindslev-Jensen C. Clinical value of component-resolved diagnostics in peanut-allergic patients. Allergy 2013; 68: Figure legends Figure 1. Evolution of sensitization (IgE 0.3 ISU-E or 0.35 ku/l, respectively) prevalence (A) and IgElevels among sensitized individuals (B) to peanut allergen molecules and peanut extract at 4, 8 and 16 years of age in a population-based study group. N=778. Footnote: 1A: proportion permanently desensitized if once sensitized to the specific allergen molecule (remission). for comparison with Ara h 8 sensitization; Sensitization rates to birch Bet v 1 were 12.6%, 17.5% and 25.8% at 4, 8 and 16 years of age, respectively B: *IgE levels to peanut allergen molecules and peanut extract did not differ significantly among sensitized individuals in the population-based study group (n=778) compared to the additional peanut extract sensitized participants (n=84). 10

12 Asarnoj et al Figure 2. Peanut and birch pollen extract sensitization overlap measured with ImmunoCAP ( 0.35 ku A /L) at 4, 8 and 16 years of age. Proportion of Ara h 2 sensitization and Ara h 8 monosensitization in the subgroups measured with the MeDALL chip ( 0.3 ISU-E). N=778 (population-based study group) Footnote: *p-values from t-test on log transformed values, not including the 0-values in each symptom subgroup 11

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23 Asarnoj et al Online Repository text Methods Study participants BAMSE is an unselected population-based birth cohort study of 4089 children 1. For this study, a questionnaire was available from baseline (2 months), 1, 2, 4, 8 and 16 years. Blood was drawn at 4, 8 and 16 years. Sera were available for 64%, 60%, and 62% of the population. In 1699 children, 42% of the original cohort, blood samples were available from all three clinical follow ups (4, 8 and 16 years). In this group of children, a random subset of 800 was collected of which 778 had complete data on peanut allergy at 16 years and sufficient serum volumes for analysis, denoted as the population-based study group. For some of the analyses, this population-based study group was enriched with all remaining peanut extract-sensitized children (measured with ImmunoCAP (f13) at 4, 8 and/or 16 years of age) among the group of This resulted in 84 additional children for which complete peanut symptom data at 16 years of age and sufficient volume of serum for analysis were available. Thus, our peanut-enriched study group included 862 children (778 from the population-based study group enriched with 84 peanut sensitized children) (Figure E1). Permission for the study was obtained from the Regional Ethical Review board at Karolinska Institutet at each follow up and parents of participating children gave their informed consent. At 16 years, the participating teens gave their separate consent. Definition of symptoms All data on peanut symptoms were collected through questionnaires answered by the children s parents at 1, 2, 4, 8 and 16 years. Specific organ symptoms to peanut were asked at 8 and 16 years. Peanut allergy at 1, 2 and 4 years: Did your child ever have adverse reactions to food or drink, such as vomiting, diarrhea, eczema, nettle rash, itch or swelling of lips or eyelids, runny nose or asthma? ( Yes and peanut indicated). 1

24 Asarnoj et al Peanut allergy at 8 years: Any of the following symptoms to peanut indicated: Nose/Eye problems, Itching in mouth, Trouble breathing, Vomiting or diarrhea, Eczema, Nettle rash, Avoided foodstuff because of previous adverse reaction. Peanut allergy at 16 years: Any of the following symptoms to peanut indicated: Difficulty breathing, asthma, cough, Itchy nose, stuffy nose, runny nose, itchy eyes, Nettle rash covering most of the body, Nettle rash that covered less, Vomiting, stomach pain, Swelling in face, eyelids, lips, Hoarseness, indistinct speech, Swollen feeling in mouth, throat, Itch in mouth, throat, ears, Pronounced fatigue, decreased awareness and Unconsciousness. Individuals with a positive answer to Itch in mouth, throat, ears and negative to all other symptoms were regarded as having local symptoms only (Oral Allergy Syndrome=OAS). Systemic peanut symptoms were defined as at least one positive answer to Difficulty of breathing, asthma, cough or Nettle rash covering most of the body or Vomiting, stomach pain or Swelling in face, eyelids, lips or Hoarseness, indistinct speech or Pronounced fatigue, decreased awareness or Unconsciousness. Allergen-specific IgE measurement Serum samples were analyzed with ImmunoCAP (Thermo Fisher AB, Uppsala, Sweden) for allergenspecific IgE antibodies to peanut extract (f13) and at 8 years of age also to Ara h 2. A positive test was defined as 0.35 ku A /L. An IgE antibody level >100 ku A /L was given the value of 101 ku A /L in statistical evaluations and an IgE level <0.35 was set to 0. IgE reactivity to the peanut allergen molecules (Ara h 1, Ara h 2, Ara h 3, Ara h 6, Ara h 8 and Ara h 9) was analyzed using a modified allergen chip based on ISAC technology (Thermo Fisher) developed in the MeDALL FP7-funded research programme 2. The cut-off was set at 0.3 ISU-E. Biases The diagnosis of peanut allergy was based on clinical symptoms and no food challenge was carried out, since the different peanut allergy phenotypes in the study population were not yet known at sampling and, for logistic reasons, it was not possible to perform food challenges in all 862 participants. Sample size 53 No sample size was calculated as this is an exploratory study. 2

25 Asarnoj et al Statistical methods Results are expressed as numbers and proportions (%). T-test of proportions was used for comparison of prevalence rates between groups. Group IgE levels are expressed as median value and range. T-test on log-transformed values was used for group comparisons of IgE levels. Odds Ratios (ORs) for symptoms to peanut at 16 years in relation to sensitization at 4 and 8 years were estimated using logistic regression models and 95% confidence intervals. Fitted predicted probability estimates were plotted according to the IgE-level (ISU-E and ku A /L) to peanut allergen molecules or peanut extract (respectively) per participant, using the results from the logistic regression. The correlation factor (rho) between the IgE levels to Ara h 2 at 8 years measured with microarray and ImmunoCAP was high: A linear regression with ImmunoCAP and microarray Ara h 2 IgE levels was performed to calculate corresponding values. 95% OR CI not including 1 and p-values <0.05 were considered significant. Analysis with logistic regression in order to investigate peanut symptoms in relation to sensitization to different peanut allergen molecules could not be performed due to substantial co-sensitization between different peanut storage proteins. All statistical analyses were performed with STATA Statistical Software (release 14.0; StataCorp, College Station, Texas, USA). References 1. Wickman M, Kull I, Pershagen G, Nordvall SL. The BAMSE project: presentation of a prospective longitudinal birth cohort study. Pediatr Allergy Immunol 2002; 13 Suppl 15: Lupinek C, Wollmann E, Baar A, Banerjee S, Breiteneder H, Broecker BM, et al. Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergenchip. Methods 2014; 66:

26 Table E1. Prevalence of reported symptoms to peanut at 4, 8 and 16 years of age. Type of symptom specified at 8 and 16 years of age. N=778 (population based). 1 year 2 years 4 years 8 years 16 years Reported symptoms to peanut Sensitization to peanut extract Peanut symptomatic and sensitized to peanut extract Sensitization to peanut storage protein Peanut symptomatic and sensitized to peanut storage proteins Peanut systemic symptom(s) and sensitized to peanut storage proteins Type of symptom* 3/769(0.4%) 6/777 (0.8%) 24/760 (3.2%) n.a. n.a. 49/778 (6.3%) n.a. n.a. 17/760 (2.2%) n.a. n.a. 44/778 (5.7%) n.a. n.a. 17/760 (2.2%) 48/773 (6.2%) 53/778 (6.8%) 63/773 (8.2%) 61/778 (7.8%) 32/768 (4.2%) 32/778 (4.1%) 42/778 (5.8%) 48/778 (6.2%) 27/773 (3.5%) 30/778 (3.9%) n.a. n.a. n.a. 11/773 (1.4%) 23/778 (3.0%) p=0.03 Upper resp n.a. n.a. n.a. 9/28 (32.1%) 6/53 (11.3%) Oral cavity n.a. n.a. n.a. 14/28 (50.0%) 37/53 (69.8%) Lower resp n.a. n.a. n.a. 11/28 (39.3%) 20/53 (37.7%) Gastrointestinal n.a. n.a. n.a. 5/28 (17.9%) 9/53 (17.0%) Urticaria n.a. n.a. n.a. 8/28 (28.6%) 13/53 (24.5%) Circulatory n.a. n.a. n.a. n.a. 1/53 (1.9%) 2 systemic symptoms n.a. n.a. n.a. 4/28 (14.3%) 16/53 (30.2%) p=0.11 n.a.= information not available resp=respiratory *only 28 of the 48 peanut symptomatic children specified type of symptom at 8 years of age.

27 Table E2. Baseline (at median age 2 months) and age one year characteristics of the populationbased study group (N=778, number of missing values in each row: 0-7) and the peanut extract sensitized enriched study group (N=862, number of missing values: 0-12), compared to children in the original cohort (N=4089, number of missing values: 0-170). Variables Study base cohort (N=4089) Study population, population based (N=778) Additional peanut extract sensitized group (N=84) n % n % p-value n % p-value Female Parental history of allergy <0.01 Breastfed 4months Parental smoking Young mother at birth ( 25 years) White collar parent Older siblings

28 E-Figure legends Figure E1. Venn diagram showing the study population of 862 participants (indicated in pink) derived from the BAMSE cohort (N=4089): A subgroup of population-based participants (N=778) enriched with peanut extract sensitized participants (N=84) from the group of eligible participants (N=1699). Figure E2. Ara h 2/Ara h 6 sensitization overlap at 4, 8 and 16 years of age. Median IgE levels (ISU-E) in the sensitization subgroups. N=862 (peanut-enriched study group) Figure E3. Ara h 1/Ara h 2/Ara h 3 sensitization overlap at 4, 8 and 16 years of age. Median IgE levels (ISU-E) in the sensitization subgroups. N=862 (peanut-enriched study group) Figure E4. Likelihood of reporting symptoms to peanut at 16 years of age in relation to A) Ara h 2 IgE level at 4 years of age (ISU-E), B) Ara h 2 IgE level at 4 years of age (ISU-E) (incident symptoms), C) peanut extract IgE level at 4 years of age (ku A /L) or D) number of sensitizing peanut allergen molecules (Ara h 8 excluded)/ peanut extract sensitization at 4 years of age. N=862 (peanut-enriched study group)